1. Field of the Invention
This invention is in the field of immunoassay devices and methods where fiber optic sensors are used.
2. The Prior Art
Optical fiber consists of transparent material such as glass or plastic. Most optical fiber is fused silica and most plastic fiber is polymethylmethacrylate (PMMA). All optical fiber consists of a core and cladding of which the core has higher refractive index than the cladding. The fiber structure guides light by the process of total internal reflection (TIR). In silica fibers the core is usually established through doping with Germanium. PMMA uses a Fluorine polymer coating as the cladding. Fibers fall into two basic types, single mode or multimode. In single mode fibers the core is very small, 5 to 10 microns in diameter, for instance. Multimode fibers have cores of 50 to several thousand microns and very small cladding (in the order of tens of microns). Single mode fibers have a large cladding (usually more than 50 microns) making the fiber diameter generally 125 microns. The purpose of the large cladding in single mode fibers is to protect and contain the evanescent field of the single mode which extends into the cladding for several microns and can contain more than 10 percent of the optical energy normally thought of as traveling only through the core. Another importance of this larger diameter cladding is so that the fibers are hot too small for handling.
Fiber optic (FO) immunosensors for immunoassays may be classified as belonging to one of several categories. In the first type of fiber optic immunoassay sensor, a fiber is stripped of its cladding. To date these types of FO sensors have all been multi-mode. The various modes strike the glass air interface and are totally internally reflected. Just on the other side of the interface, where the reflection occurs, the light exists for a short distance in the physical form of an evanescent field. If a monoclonal or polyclonal antibody is attached to this surface, the field permeates this layer of antibody molecule but, there is little or no absorption or other phenomena which would otherwise change the amount of the light contained within the fiber. When antigens which have been labeled with fluorophores are attached, the evanescent field can cause the antigens to fluoresce causing an emittance which is optically detectable in a reduction in the light level or through collection of the fluorescence. In this way light energy is taken from the core through the evanescent field and is used to cause fluorescence. It is usually necessary to conjugate a fluorophore to the antigen to accomplish this application. If no antigen is bound, no fluorescent Output occurs. In some cases it is the unknown antigens which are attached and the antibodies are allowed to bind, assuming there is specificity to the fluorescent antibodies. The sensor can detect antigens or antigens and can be bound and the biosensor used to detect the presence of antibody (as in a disease state such as following infection with HIV or tuberculosis).
The fiber optic sensor can possibly replace many other immunodiagnostic modalities currently available. By binding the antigen, the sensor can probe unknown antibody. By binding an antibody, the assay can detect specific antigenic toxins or other immunogenic targets. Thus an organism is suspected to have been producing as a result of contact with an infectious agent.
The second type of fiber optic immunoasay sensor uses a coating deposited onto a fiber tip which may be illuminated with pulse which in turn induces fluorescence which is reflected back up the fiber and later detected. A beam splitter and possibly a filter is usually used to separate the pulses from the fluorescence. As in the first type, either antibody or antigen sensors may be constructed. Several options are available for tip and geometries. Review articles are available in Volume I and II of Fiber Optic Chemical Sensors and Biosensors, Otto S. Wolfbeis, ed., CRC Press, Boca Raton (1991). A specifically relevant article appears in Volume II, Vo-Dinh, et al., "Fiberoptics Immunosensors," Chapter 17, page 271-257.
A third type of fiber optic immunoassay sensor involves a stripped fiber which has antibodies or antigens attached to the core-air interface. This sensor, see R. G. Heideman, et al., "Simple interferometer for the evanescent field refractive index sensing as a feasibility study for an immunosensor," Applied Optics, Vol. 20, No. 12, pages 1474-1749 (1991), is used as one leg of a fiber optic Mach-Zehnder interferometer. The binding of molecules to the surface during attachment of either antibody or antigen suffices to locally change the index of refraction at the core solution interface. This occurs because the propagation constant of the cladding sets the speed of light within the core and some of this energy is in the cladding as an evanescent field, where the index is raised. Then, the phase velocity of the light changes and interference fringes are detected at the output of the interferometer.
Still other configurations of immunosensors have been described such as surface plasmon resonance immunosensors, and grating couplers used as integrated optical chemical sensors. These and others are discussed in the book edited by Wolfbels, op. cit..
The first type of fiber optic immunoasay sensors are fiber optic variations of internal reflection spectroscopy technology and are thoroughly described in Internal Reflection Spectroscopy by N. J. Harrick, Harrick Scientific Corporation, Ossining, N.Y. (1987). Harrick describes all kinds of geometries for these multimode sensors. A reference for the theory of fiber optics is Optical Wave Guide Theory by a W. Snyder and J. D. Love, Chapman and Hall, New York (1983). See also G. Stewart, et al., "Chemical sensing by evanescent field adsorption: the sensitivity of optical waveguides", "Proceedings of the international Society of Optical Engineering (SPIE)", Vol. 990, Boston,1988.
The evanescent field extends for only about one optical wavelength beyond the actual fusion joint surface. For light of 830 nm wavelength, it is not necessary to coat the fibers to more than one micron in thickness.
The advantages of coating single mode couplers are many. Conventional evanescent wave spectroscopy has been thoroughly described by N. J. Harrick, op. cit.. These are the techniques also used today in fiber optic sensing. The processes of detection usually rely on absorption processes in regions of a slab or waveguide where the evanescent field has penetrated the guide. In contrast, the single mode coupler sensor approach has evanescent field everywhere along the fusion joint. The guides are arranged so that as many reflections occur as possible. This is done by launching coherent light into the guide at angles which favor multiple reflections. Even so the numbers of places where ray optics allows interaction between the optical field and the chemical species are very few. The same thing happens in large core optical fibers; there is very little interaction area for the total area available. These sensors are only useful at the site where TIR takes place and so multiple reflections are used to amplify the effect. Even with favorable geometries the area of sensor surface which supports evanescent fields is small compared to the whole surface area.
Another problem with large core fibers is related to the relatively large number of spatial modes supported, possibly in the hundreds. The modes can and do interfere with each other leading on one hand to a finite number of modes and on the other hand to a speckle pattern occurring at the detector Snyder and Love, Optical Waveguide Theory, Chapman and Hall, N.Y. 1983. The detector can't distinguish the variations in light levels due to absorption from the many types of perturbations which cause speckle.
In contradistinction, single mode fibers only support the lowest order spatial mode and there is no speckle in these systems. Coupler sensors also are not subject to modal redistribution due to environmental effects. Another significant advantage of single mode fibers is that the evanescent field surrounds the entire space immediately surrounding the core. Couplers can locally force more than 90% of the optical energy into the evanescent field. It should be noted, however, that since single mode waveguides may not be analyzed using the ray optic approximation, electromagnetic waveguide theory must be applied.
SUMMARY OF THE INVENTION
This invention provides a sensitive detector having a wide variety of applications in the fields of biology, biochemistry and chemistry, and in many clinical applications as well. It uses a fiber optic coupler output ratio change due to the chemical/biochemical/bioaffinity/immunogenic-type interaction of bio-molecules (ligands) with their respective binding partners. The terms ligand and its binding partner for the ligand or, simply, binder will be used to represent the two components in specific bioaffinity binding pairs, all of which are capable of recognizing and binding with the other partner in a bimolecular recognition pair. Examples of such binding pairs include: antigen-antibody, substrate-enzyme, effector-enzyme, inhibitor-enzyme, complementary nucleic acid strands, binding protein-vitamin, binding protein-nucleic acid; reactive dye-protein, reactive dye-nucleic acid; and others. Either of the bindings partners is permanently attached to the fusion joint of a single mode coupler and within the evanescent field for very specific and direct detection of minute concentrations of an analyte of interest (one of the components of the binding pair) within the tested sample without the need to label a reagent. The biomolecule may be linked to the surface of the fusion joint by means of a spacer molecule.
In the following the invention will occasionally be specifically described with reference to immunosensors, it being understood that the invention is in no way confined thereto and covers quite generally chemical sensors within the meaning of this term as defined hereinabove. In particular, beyond immunosensors, this invention has an application to an additional field of chemistry which is binding of nucleotides, cDNAs, riboprobes, genetic DNA or RNA molecules using the principles of solution hybridization. This expands the scope of the immunosensor to a "nucleotide sensor" with potential for wide applications in many aspects of the field of molecular biology. The binding of single stranded synthetic oligonucleotides (or cDNA or genomic DNAs) to the fiber optic will allow for the detection of the complimentary DNA strand or RNA. This application has potential uses in many areas: 1) the evaluation of levels of mRNA expression in tissues, e.g., the determination of tissue specificity of gene expression; 2) the detection of viral or other rare DNAs; 3) forensic science, paternity testing. The sensor may also substitute for Northern or Southern or Dot blots in some instances.
Construction of conventional single mode evanescent wave spectrometer systems involves stripping or exposing the fiber core by removal of the fiber's protective cladding. As in conventional evanescent wave spectroscopy, there is an absorption of light at some particular wavelength and, as a result, the detector experiences a lowering of the optical intensity level as light is extracted at the exposed core section of the fiber. It should be appreciated that optical power levels are extremely difficult to measure in absolute terms; the best photometers commonly available are only accurate to about 1%. Relative measurements may be measured with much more accuracy but longer term measurements are subject to drift. Signal drift occurs at the detector, in the transimpedance amplifiers behind the detector, at the laser source and it's driving electronics, and even in the fiber and connectors. Signal drift is difficult to separate from sensor activity without some kind of a reference.
Coupler sensors with a single mode, share the advantages of the conventional single mode sensors. However, absorption is not necessary and is not even desirable for the operation of the sensor. As the coupler output ratio is extremely sensitive to the refractive index of the medium which surrounds the fusion joint of the coupler, chemical changes occurring near the fusion joint will cause a trading of power between the output channels. Changes in refractive index of one part in a million have been measured with the simple hand-held optical power meters used in fiber optics communication systems.
Coupler sensors provide a signal processing advantage which is important to the measurement of small signal changes. Since there is no signal lost to absorption, the two coupler output channels will sum to a constant level even though the coupling ratio may change drastically. The dual output provides a total separation of drift variables from sensor variables. Difference over sum signal processing is also available as are a number of attractive electronic methods common to self referenced systems. Thus in view of this consideration in the construction of antibody conjugated optical fiber couplers may provide useful probes for detecting soluble ligands and similarly, the construction of nucleotide sensors, with conjugate DNA or RNA, may provide improved methods for detection of complimentary DNA or RNA.
The development of coupler fabrication technologies now provide an electric furnace method of biconical coupler fabrication. A coupler fusion electric furnace taught by U.S. Pat. No. 4,879,454, Gerdt, (incorporated herein by reference) allows inexpensive production of single mode couplers.
The remarkable sensitivity of coupler sensors has been documented, see D. W. Gerdt, L. H. Gilligan, "Variable Coupler Fiber Optic Sensor", Proceedings of the international Society of Optical Engineering (SPIE), Vol. 835, 1987, D. W. Gerdt, "Applications of Fiber Optic Coupler Sensors,"Proceedings of the International Society of Optical Engineering (SPIE), Vol. 990 Boston, 1988, and D Gerdt, "Fiber Optic Coupler Sensors," Naval Eng. Journal, May 1990, 275-280). Coupler sensors can be used in an assay method for very dilute, specific antigens, e.g., proteins including glycoproteins. The attachment of polyclonal antibodies can be used as screening tests for one or more of several specific antigens. The coupler output ratio depends very strongly on the refractive Small changes in index change greatly or even switch totally the division of optical power between the output fibers.
An antibody which is immobilized on the surface of the fusion joint can be used as an assay for a specific antigen. The basic concept of the fiber optic coupler also applies to DNA/RNA chemistries as well, provided conjugation methods to the couplers are developed. A coupler-antibody system can be prepared with an antibody and then exposed to a test solution containing an antigen capable of binding with this antibody. Upon first contact the coupler output ratio will immediately change to a certain value. This value will remain constant if no specific antigens are present. As the reaction proceeds, the antigen capable of binding with the antibody, if present, will begin to bind to the antibody until the degree of binding reaches an equilibrium value after a few minutes. The binding will occur only within the evanescent field of the coupler where the output ratio of the coupler is determined. Binding changes dramatically the electronic distribution of the original fusion joint coating because the index of refraction depends almost entirely on electronic arrangement and densities.
The rate at which the coupling ratio changes depends on the concentration of the analyte in solution. A coupler sensor used in this manner acts as an antibody immobilized on a solid support in an immunoassay.